WB smears were stained with selleck chem a hematoxylin-eosin-based rapid stain (Pan��tico r��pido, Laborclin, Brazil) and observed by microscopy (100X objective, under immersion oil). The M- and G-enriched samples were obtained from 4mL of WB with the SepCell kit (LGC Biotecnologia, Brazil), according to the manufacturer’s instructions. The BC fraction was collected from 1mL blood that was centrifuged at 12,000g for 10min.2.3. DNA ExtractionFrom each dog, a sample of blood was collected, and the DNA was extracted. Four milliliters of blood were extracted with sodium citrate and 1mL without sodium citrate. The DNA samples from the WB (200��L), BC (50��L), M (50��L), G (100��L), and C (50��L) fractions were extracted with a commercial kit (Invisorb Spin Blood Midi kit; INVITEK), following the manufacturer’s instructions.

The DNA from 21 WB, 19 G and 19M, 20 BC, and 15 C samples was used in the nPCR to amplify the E. canis and A. platys 16S rRNA sequences.2.4. Nested PCR (nPCR)The first round of PCR used 0.5 to 1.0��g of the genomic DNA, and the primers ECC and ECB were designed to amplify a 478 base-pair (bp) fragment of the Ehrlichia 16S rRNA [13]. The second round of PCR used a 1.0��L aliquot of the first reaction as a template and the EHCA sense/EHCA antisense [14] and EHPL sense/EHPL antisense (Jo?o Pessoa Ara��jo Jr.: pers. comm., 2010) primers, which were designed to amplify a 389bp fragment for E. canis and 384bp fragment for A. platys, respectively. Separate reactions were used to detect each species individually. The primers are described in Table 1.

The primer design was confirmed with the software Primer 3 (http://fokker.wi.mit.edu/primer3/input.htm). The reaction mix contained 1X reaction buffer (50mM KCl, 20mM Tris-HCl (pH 8.4), and 0.1% Triton X-100), 1.75mM MgCl2, 0.2mM dNTP mix, 1��M PCR primers, 0.625U Taq DNA polymerase, and autoclaved ultrapure water to a final volume of 25��L. The thermocycle was as follows: 94��C for 10 minutes followed by 40 cycles at 94��C for 60 seconds, 60��C for 60 seconds, 72��C for 60 seconds, and a final step of 72��C for 4 minutes before holding at 4��C. Ultra-pure autoclaved water was used as negative control in each PCR batch. The genomic DNA from confirmed E. canis and A. platys cases was used as positive controls for the E. canis 16S rRNA and A. platys 16S rRNA genes, respectively. Ten microliters of the final products were electrophoresed at 90 volts for approximately 1 hour in 1.5% agarose gels containing ethidium bromide in Drug_discovery Tris-Borate EDTA (TBE). The E. canis and A. platys reactions were positive when a 389 or a 384bp fragment was detected, respectively.Table 1The primer sequences for the 16S rRNA gene used to detect the E. canis and A. platys by the nPCR reactions.2.5.